Regulator/promoter for tunable gene expression and metabolite sensing

ABSTRACT

A method for the facile and inexpensive inducible expression of heterologous genes has been discovered. The yhcS regulator gene has been found to be inducible by aromatic carboxylic acids and to alter the expression of operons in the LysR gene family, including the yhcRQP operon, common in enteric bacteria. Heterologous nucleic acid molecules placed under the control of yhcs responsive promoters may be overexpressed in response to the presence of inexpensive aromatic carboxylic acids.

This application claims the benefit of U.S. Provisional Application No.60/440,965 filed Jan. 17, 2003, which is hereby incorporated in itsentirety by reference.

FIELD OF INVENTION

The present invention relates to the fields of molecular biology andmicrobiology. More specifically, this invention pertains to a novelsystem for controlling gene expression levels that is induced byinexpensive, environmentally friendly, small molecules.

BACKGROUND

There is a need in the field of microbial metabolic engineering fortunable promoters and novel regulatory switches for the inducibleexpression of heterologous proteins. The “old standard” lac promoter/Lacrepressor system is still widely used. However, the commonly usedinducer, isopropylthio-beta-D-galactoside (“IPTG”), is expensive andthus not practical for large-scale bioprocesses. Additionally, higherconcentrations of IPTG increase the metabolic burden on the cell, inturn reducing the maximal expression of the target gene (Donovan et al.,J. Ind. Microbiol. 16:145-154 (1996)). The few available alternativesalso have limitations.

One possible solution to this problem is to appropriate existing geneticsystems of transcriptional regulators to enhance heterologous geneexpression. A number a of transcriptional regulators are known. Forexample, the LysR family of transcriptional regulators is one of thelargest groups of transcriptional regulators in prokaryotes (Schell,Annu. Rev. Microbiol. 47:597-626 (1993)). Currently, there are over 80known members of this regulator family. Proteins having greater than 20%amino acid identity with another LysR family member or having theconsensus sequence of the N-terminal region of the LysR family areconsidered to be members of this regulator family. LysR family membersare also commonly found in the size range of 276 to 324 amino acids,bind to similar DNA sequences in the absence of inducers, have promotersthat are located close to or overlapping those of the regulated targetgene, and most can repress their own transcriptional levels 3- to10-fold. Activation of the regulated target gene occurs in the presenceof inducer and usually results in a 6- to 200-fold increase in regulatedtarget gene transcription. Regulated target genes are diverse and havenumerous functions.

Recently, the gene encoded by open reading frame (“ORF”) b3243 inEscherichia coli (“E. coli”) has been demonstrated to function viaquorum sensing (Sperandio et al., Infect. Immun. 70:3085-3093 (2002)).Quorum sensing is the ability of bacteria cells to communicate with oneanother through perception of the accumulation of signaling moleculesbased on bacteria cell density. The gene encoded by ORF b3243 isup-regulated via quorum sensing resulting in a 23-fold increase intranscription of the gene. The protein produced by the b3243 ORF was notpurified. The gene was found to have a role in the regulation of the LEEgenes involved in a type III secretion system, a pathogenecity systemthat serves to translocate, upon contact with eukaryotic host cells,proteins from the bacteria cytoplasm into the host cell cytoplasm. Asthe b3243 ORF gene is a putative regulator of the LysR family, the b3243ORF gene itself was able to induce a four-fold induction of LEE1transcription. No inducer of the b3243 ORF gene was identified. One inthe art will appreciate that this inefficient induction prevents theb3243 ORF gene from being a viable promoter/regulator system without theidentification of its inducer.

The use of promoter/reporter gene constructs is well known in the art(Serebriiskii and Golemis, Anal. Biochem. 285:1-15 (2000); Spergel etal., Prog. Neurobiol. 63:673-686 (2001); Yarranton, Curr. Opin.Biotechnol. 3:506-511 (1992)). Particularly, reporter systems utilizingβ-galactosidase, green florescent protein, luciferase, andchloramphenicol acetyl transferase (CAT) are all commonly used in theart. Additionally, reporter systems such as luxCDABE are useful becausethis operon contains all of the genes required for bioluminescentreporting ((Van Dyk et al., Proc. Nat. Acad. Sci. USA 98:2555-2560(2001)).

The luxCDABE operon has been utilized to create a collection of randomgene fusions, comprising 27% of the known or predicted transcriptionalunits of E. coli (Van Dyk et al., J. Bacteriol. 183:5496-5505 (2001)).Treatment of E. coli cells containing these gene fusions with nalidixicacid, a quinolone, results in selective up-regulation of ten genes. Someof these up-regulated genes are LexA-regulated SOS genes, while othersare not generally induced by DNA damage.

Aromatic compounds such as aromatic carboxylic acids are usually toxicto microorganisms. Numerous bacterial strains resistant to aromaticcompounds, however, are known in the prior art (Diaz et al., Microbiol.Mol. Biol. Rev. 65:523-569 (2001)). Additionally, a LysR family memberfrom Acinetobacter, BenM, is responsive to synergistic induction bybenzoic acid, an aromatic carboxylic acid, and muconic acid (Bundy etal., Proc. Natl. Acad. Sci. USA 99:7693-7698 (2002)). Benzoic acidalone, however, produces minimal, if any, induction of BenM activity.Even the synergistic response with muconic acid produces only afour-fold increase in BenM activity.

U.S. Pat. No. 5,292,643 issued to Shibano et al. on Mar. 8, 1994describes genes related to fusaric acid resistance in variety ofmicroorganisms. Specifically, genes capable of decomposing ordetoxifying fusaric acid are disclosed. One of the genes postulated tobe involved in fusaric acid resistance, fusB, shares some homology withthe putativTe efflux transporter (PET) yhcP gene (Paulsen et al., FEMSMicrobiol. Lett. 156:1-8 (1997)). Applicants incorporate by referencethe co-owned and concurrently filed application entitled “PET Family ofEfflux Proteins”, U.S. Patent Application No. 60/440,760, whichdescribes new proteins efflux proteins whose expression may alter theexpression of carboxylic acids.

The problem to be solved therefore is to discover facile and inexpensivemethods of inducible expression of heterologous genes. Applicants havesolved the stated problem through the discovery that the promoterelements of the yhcRQP operon are responsive to the expression of theyhcS regulator (a member of the LysR family of transcriptionalregulators) whose expression may be induced by an inexpensive cadre ofaromatic carboxylic acid inducers.

SUMMARY OF THE INVENTION

The invention relates to the discovery that the yhcS regulator isresponsible for the activation of the yhcRQR operon promoter and isinducible by aromatic carboxylic acids, compounds that are typicallytoxic to cells. The expression of heterologous genes, placed under thecontrol of a yhcRQP operon promoter may be regulated by the presence ofan aromatic carboxylic acid. Accordingly the invention provides a methodfor the inducible expression of a heterologous nucleic acid moleculecomprising:

-   a) providing a host cell having a genome comprising:    -   i) a yhcS regulator gene responsive to an aromatic carboxylic        acid inducer;    -   ii) a promoter region, responsive to expression of the yhcS        regulator gene; and    -   iii) at least one heterologous nucleic acid molecule;-    wherein the at least one heterologous nucleic acid molecule is    operably linked to the promoter region;-   b) contacting the host cell of (a) with an aromatic carboxylic acid    inducer wherein the at least one heterologous nucleic acid molecule    is expressed.

In a preferred embodiment the invention provides a method for theinducible expression of a heterologous nucleic acid molecule comprising:

-   a) providing an enteric bacterial host cell having a genome    comprising:    -   i) a yhcS regulator gene responsive to an aromatic carboxylic        acid inducer;    -   ii) a promoter region, responsive to expression of the yhcS        regulator gene; and    -   iii) at least one heterologous nucleic acid molecule;-    wherein the at least one heterologous nucleic acid molecule is    operably linked to the promoter region;-   b) contacting the host cell of (a) with an aromatic carboxylic acid    inducer wherein the at least one heterologous nucleic acid molecule    is expressed.

Specific yhcS regulator gene useful in the present invention are thosethat are selected from the group consisting of:

-   -   a) an isolated nucleic acid molecule comprising nucleic acid        sequence SEQ ID NO:1; and    -   b) an isolated nucleic acid molecule, which hybridizes to SEQ ID        NO:1 after being washed with 0.1×SSC, 0.1% SDS at 65° C. and        washed with 2×SSC, 0.1% SDS followed by a second wash in        0.2×SSC, 0.1% SDS.

Similarly specific promoter responsive to expression of the yhcSregulator gene are those selected from the group consisting of:

-   -   a) an isolated nucleic acid molecule comprising nucleic acid        sequence SEQ ID NO:3; and    -   b) an isolated nucleic acid molecule, which hybridizes to SEQ ID        NO:3 after being washed with 0.1×SSC, 0.1% SDS at 65° C. and        washed with 2×SSC, 0.1% SDS followed by a second wash in        0.2×SSC, 0.1% SDS.

In another embodiment the invention provides a host cell comprising:

-   a) a yhcS regulator gene responsive to an aromatic carboxylic acid    inducer having a nucleic acid sequence selected from the group    consisting of:    -   i) an isolated nucleic acid molecule comprising nucleic acid        sequence SEQ ID NO:1; and    -   ii) an isolated nucleic acid molecule, which hybridizes to SEQ        ID NO:1 after being washed with 0.1×SSC, 0.1% SDS at 65° C. and        washed with 2×SSC, 0.1% SDS followed by a second wash in        0.2×SSC, 0.1% SDS;-   b) a promoter region, responsive to expression of the yhcS regulator    gene having a nucleic acid sequence selected from the group    consisting of:    -   i) an isolated nucleic acid molecule comprising nucleic acid        sequence SEQ ID NO:3; and    -   ii) an isolated nucleic acid molecule, which hybridizes to SEQ        ID NO:3 after being washed with 0.1 ×SSC, 0.1% SDS at 65° C. and        washed with 2×SSC, 0.1% SDS followed by a second wash in        0.2×SSC, 0.1% SDS; and    -   iii) at least one heterologous nucleic acid molecule;-    wherein the at least one heterologous nucleic acid molecule is    operably linked to the promoter region.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detaileddescription, figures and the accompanying sequence descriptions, whichform a part of this application.

FIG. 1 shows the kinetics of the yhcRQP-luxCDABE response to pHBA inyhcS⁺ and yhcS⁻ host strains.

The following sequences conform with 37 C.F.R. 1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) andconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

SEQ ID NO:1 is the nucleotide sequence of the yhcS regulator.

SEQ ID NO:2 is the amino acid sequence of the YhcS protein.

SEQ ID NO:3 is the nucleotide sequence of the promoter region upstreamof yhcQ.

SEQ ID NO:4 is the nucleotide sequence of the primer Kan-2FP(PCR).

SEQ ID NO:5 is the nucleotide sequence of the primer Kan-2RP(PCR).

SEQ ID NO:6 is the nucleotide sequence of the primer Kan-2FP-1.

SEQ ID NO:7 is the nucleotide sequence of the primer Kan-2RP-1.

SEQ ID NO:8 is the nucleotide sequence of the primer YhcS.F.

SEQ ID NO:9 is the nucleotide sequence of the primer YhcS.R.

DETAILED DESCRIPTION OF THE INVENTION

There is a need in the field of microbial metabolic engineering fortunable promoters and novel regulatory switches. Advantages ofApplicants' system are the very low basal levels of gene expression inthe absence of inducer and expression levels that vary with theconcentration of inducer up to relatively high expression levels.Furthermore, the cost of many of the inducing molecules is relativelyinexpensive.

There are numerous uses for Applicants' tunable promoter systemcomprising the YhcS regulatory protein and the responsive promoterregion. This novel promoter/regulator system can be used to control andregulate expression of genes and operons of interest by applyingstandard molecular biology methods as has been demonstrated herein bythe controlled expression of luxCDABE.

Another possible application is suggested by analogy to other LysRfamily proteins, which typically have a conformational change uponbinding their cognate inducer. YhcS protein likely binds to certainaromatic carboxylic acid molecules and changes conformation such that itactivates gene expression upon DNA binding. This conformation change maybe useful to sense small molecules in applications such as those foundin nano scale systems.

The range of molecules to which YhcS responds may be manipulated byapplication of a variety of protein engineering techniques. Thus, theuseful range of molecules of each of the above-mentioned applicationscould be expanded.

Advantages of this regulator/promoter system include, but are notlimited to, basal expression levels that are extremely low; high levelsof expression following induction; inexpensive, environmentally friendlyinducers; when used in conjunction with other expression systems, analternative expression system that will allow differential regulation ofvarious genes in a genetically engineered host; and a proteinconformation change useful for nanotechnology.

Applicants specifically incorporate the entire content of all citedreferences in this disclosure.

In the context of this disclosure, a number of terms shall be utilized.

The term “pHBA” is the abbreviation for para-hydroxybenzoic acid, whichis also known as para-hydroxybenzoate.

The term “pHCA” is the abbreviation for para-hydroxycinnamic acid, whichis also known as para-hydroxycinnamate.

The term “CA” is the abbreviation for cinnamic acid, which is also knownas cinnamate

An “isolated nucleic acid molecule” refers to a polymer of RNA or DNAthat is single- or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases. An isolated nucleic acidmolecule in the form of a polymer of DNA may be comprised of one or moresegments of cDNA, genomic DNA or synthetic DNA.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength. Hybridization and washing conditions are well known andexemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989), particularly Chapter11 and Table 11.1 therein (entirely incorporated herein by reference)(hereinafter “Sambrook”). The conditions of temperature and ionicstrength determine the “stringency” of the hybridization. Stringencyconditions can be adjusted to screen for moderately similar fragments,such as homologous sequences from distantly related organisms, to highlysimilar fragments, such as genes that duplicate functional enzymes fromclosely related organisms. Post-hybridization washes determinestringency conditions. One set of preferred conditions uses a series ofwashes starting with 6×SSC, 0.5% SDS at room temperature for 15 min,then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and thenrepeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A morepreferred set of stringent conditions uses higher temperatures in whichthe washes are identical to those above except for the temperature ofthe final two 30 min washes in 0.2×SSC, 0.5% SDS is increased to 60° C.Another preferred set of highly stringent conditions uses two finalwashes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringentconditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washedwith 2×SSC, 0.1% SDS followed by a second wash in 0.2×SSC, 0.1% SDS, forexample.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (Sambrooksupra). For hybridizations with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (Sambrooksupra). In one embodiment the length for a hybridizable nucleic acid isat least about 10 nucleotides. Preferably, a minimum length for ahybridizable nucleic acid is at least about 15 nucleotides; morepreferably at least about 20 nucleotides; and most preferably the lengthis at least 30 nucleotides. Furthermore, the skilled artisan willrecognize that the temperature and wash solution salt concentration maybe adjusted as necessary according to factors such as length of theprobe.

A “substantial portion” refers to an amino acid or nucleotide sequencewhich comprises enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to afford putative identification ofthat polypeptide or gene, either by manual evaluation of the sequence byone skilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Basic Local AlignmentSearch Tool; Altschul et al., J. Mol. Biol. 215:403-410 (1993). Ingeneral, a sequence of ten or more contiguous amino acids or thirty ormore nucleotides is necessary in order to putatively identify apolypeptide or nucleic acid sequence as homologous to a known protein orgene. Moreover, with respect to nucleotide sequences, gene specificoligonucleotide probes comprising 20-30 contiguous nucleotides may beused in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12-15 bases may be used as amplification primers inPCR in order to obtain a particular nucleic acid molecule comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises enough of the sequence to afford specific identificationand/or isolation of a nucleic acid molecule comprising the sequence. Theinstant specification teaches partial or complete amino acid andnucleotide sequences encoding one or more particular bacterial proteins.The skilled artisan, having the benefit of the sequences as reportedherein, may now use all or a substantial portion of the disclosedsequences for the purpose known to those skilled in the art.Accordingly; the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

The term “complementary” describes the relationship between nucleotidebases that are capable to hybridizing to one another. For example, withrespect to DNA, adenosine is complementary to thymine and cytosine iscomplementary to guanine. Accordingly, the instant invention alsoincludes isolated nucleic acid molecules that are complementary to thecomplete sequences as reported in the accompanying Sequence Listing aswell as those substantially similar nucleic acid sequences.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NewJersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G.,ed.) Academic Press, New York (1987); and Sequence Analysis Primer(Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include, but are not limited to, the GCG Pileupprogram found in the GCG program package, using the Needleman and Wunschalgorithm with their standard default values of gap creation penalty=12and gap extension penalty=4 (Devereux et al., Nucleic Acids Res.12:387-395 (1984)), BLASTP, BLASTN, and FASTA (Pearson et al, Proc.Natl. Acad. Sci. USA 85:2444-2448 (1988). The BLASTX program is publiclyavailable from NCBI and other sources (BLAST Manual, Altschul et al.,Natl. Cent. Biotechnol. Inf., Natl. Library Med. (NCBI NLM) NIH,Bethesda, Md. 20894; Altschul et al., J. Mol. Biol. 215:403-410 (1990);Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). Anotherpreferred method to determine percent identity is by the method ofDNASTAR protein alignment protocol using the Jotun-Hein algorithm (Heinet al., Meth. Enzymol. 183:626-645 (1990)). Default parameters for theJotun-Hein method for alignments are: for multiple alignments, gappenalty=11, gap length penalty=3; for pairwise alignments ktuple=6. Asan illustration, by a polynucleotide having a nucleotide sequence havingat least, for example, 95% “identity” to a reference nucleotide sequenceit is intended that the nucleotide sequence of the polynucleotide isidentical to the reference sequence except that the polynucleotidesequence may include up to five point mutations per each 100 nucleotidesof the reference nucleotide sequence. In other words, to obtain apolynucleotide having a nucleotide sequence at least 95% identical to areference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with another nucleotideor a number of nucleotides up to 5% of the total nucleotides in thereference sequence may be inserted into the reference sequence. Thesemutations of the reference sequence may occur at the 5′ or 3′ terminalpositions of the reference nucleotide sequence or anywhere between thoseterminal positions, interspersed either individually among nucleotidesin the reference sequence or in one or more contiguous groups within thereference sequence. Analogously, by a polypeptide having an amino acidsequence having at least, for example, 95% identity to a reference aminoacid sequence is intended that the amino acid sequence of thepolypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid. In other words, toobtain a polypeptide having an amino acid sequence at least 95%identical to a reference amino acid sequence, up to 5% of the amino acidresidues in the reference sequence may be deleted or substituted withanother amino acid, or a number of amino acids up to 5% of the totalamino acid residues in the reference sequence may be inserted into thereference sequence. These alterations of the reference sequence mayoccur at the amino or carboxy-terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

The term “percent homology” refers to the extent of amino acid sequenceidentity between polypeptides. When a first amino acid sequence isidentical to a second amino acid sequence, then the first and secondamino acid sequences exhibit 100% homology. The homology between any twopolypeptides is a direct function of the total number of matching aminoacids at a given position in either sequence, e.g., if half of the totalnumber of amino acids in either of the two sequences is the same thenthe two sequences are said to exhibit 50% homology.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments that are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

“Gene” refers to a nucleic acid molecule that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature.

“Genome” refers to the entire genetic information contained within anorganism (e.g., chromosome, plasmid, plastid, or mitochondrial DNA).“Endogenous gene” refers to a native gene in its natural location in thegenome of an organism. A “foreign” gene refers to a gene not normallyfound in the host organism, but that is introduced into the hostorganism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure. “Structural gene” refers to a gene that codes for the aminoacid sequence of a protein or for a ribosomal RNA or transfer RNA. An“operon” refers to a controllable unit of transcription consisting of anumber of structural genes transcribed together.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Suitable regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

“Heterologous” as used in the context of gene expression relates to thatwhich is “foreign” to a particular environment. Thus, a “heterologousgene” or “heterologous nucleic acid molecule” means a nucleic acidmolecule that is foreign, or non-native to a particular host or genome.A “heterologous protein” is a protein that is foreign to a host cell andis typically encoded by a heterologous gene. Heterologous nucleic acidsof the invention are typically expressed under the control of regulatedpromoters in an inducible fashion.

The term “regulator” refers to a protein whose primary function is tocontrol the rate of expression of regulated genes. Regulation of geneexpression can be by positive activation or by repression. A “regulatorygene” refers to a gene that encodes a regulator. Within the context ofte present invention a typical regulator gene is the yhcS gene

“Host cell” refers to a cell into which has been introduced (e.g.,transformed or transfected) an exogenous polynucleotide sequence, i.e. aheterologogus nucleic acid molecule. Host cells are typicallyprokaryotic cells such as bacteria, e.g., E. coli, and may be eukaryoticcells such as yeast, insect, amphibian, green plant, or mammalian cells,where the relevant regulator genes exist.

“Inducer” refers to a small molecule that initiates transcription, orincreases the rate of transcription, of a desired gene. Within thecontext of the present invention typical inducers are aromaticcarboxylic acids having the ability to activate a regulator gene.

“Reporter gene” refers to a gene that encodes an easily assayed product(e.g. luxCDABE, bgaB, cat, dsRed, galK, gfp, lacZ, luc, luxAB, nptll,phoA, uidA, or xylE). Typically reporters are coupled to the promoterand/or regulator sequence of another gene and transfected into a hostcell. The reporter gene can then be used to see which factors activateresponse elements in the upstream region of the gene of interest.

“Translation leader sequence” refers to a DNA sequence located betweenthe promoter sequence of a gene and the coding sequence. The translationleader sequence is present in the fully processed mRNA upstream of thetranslation start sequence. The translation leader sequence may affectprocessing of the primary transcript to mRNA, mRNA stability ortranslation efficiency. Examples of translation leader sequences havebeen described (Turner et al., Mol. Biotechnol. 3:225 (1995)).

“3′ non-coding sequences” refer to DNA sequences located downstream of acoding sequence and include polyadenylation recognition sequences andother sequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor. The use of different 3′ non-codingsequences is exemplified by lngelbrecht et al., Plant Cell 1:671-680(1989).

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065).The complementarity of an antisense RNA may be with any part of thespecific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, introns, or the coding sequence. “Functional RNA”refers to antisense RNA, ribozyme RNA, or other RNA that is nottranslated yet and has an effect on cellular processes.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid molecule so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it affects the expression of that codingsequence (i.e., that the coding sequence is under the transcriptionalcontrol of the promoter). Coding sequences can be operably linked toregulatory sequences in sense or antisense orientation.

The term “expression” refers to the transcription and stableaccumulation of sense (mRNA) or antisense RNA derived from the nucleicacid molecule of the invention. Expression may also refer to translationof mRNA into a polypeptide. “Antisense inhibition” refers to theproduction of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or nontransformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020).

“Transformation” refers to the transfer of a nucleic acid molecule intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes that are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

“PCR” or “polymerase chain reaction” is a technique used for theamplification of specific DNA segments (U.S. Pat. Nos. 4,683,195 and4,800,159).

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook, J., Fritsch, E. F.and Maniatis, T., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989) (hereinafter “Sambrook”); and by Silhavy, T. J., Bennan, M. L.and Enquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory Cold Press Spring Harbor, N.Y. (1984); and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-lnterscience (1987).

The present invention relates to the discovery that aromatic carboxylicacids induce the up-regulation or expression of the regulator gene yhcSwhich in turn is responsible for the activation of the promoter regiondriving expression of the yhcRQP operon. Expression of heterologousnucleic acid molecules which are operably to these promoters maytherefore be inducibly regulated by the presence or absence ofinexpensive aromatic carboxylic acids in the medium.

Regulator Systems

The yhcS regulator and the corresponding yhcRQP operon is known and iscommon in a variety of enteric bacteria such as Escherichia (Hayashi etal., “Complete genome sequence of enterohemorrhagic Escherichia coliO157:H7 and genomic comparison with a laboratory strain K-12”, DNA Res.8 (1), 11-22 (2001)); Yersinia (Parkhill et al., “Genome sequence ofYersinia pestis, the causative agent of plague”, Nature 413 (6855),523-527 (2001)); Shigella (Wei et al., “Complete Genome Sequence andComparative Genomics of Shigella flexneri Serotype 2a Strain 2457T”,Infect. Immun. 71 (5), 2775-2786 (2003)); and Salmonella (McClelland etal., “Complete genome sequence of Salmonella enterica serovarTyphimurium LT2”; Nature 413 (6858), 852-856 (2001)). It will beappreciated by one of skill in the art that those organisms havinghomologs to the present regulators will be expected to function inheterologous gene expression in similar ways.

Those cells having existing homologous regulator systems may be used inthe present invention for the expression of heterologous DNA simply bythe insertion of the DNA to be expressed in the appropriate position inthe genome and in the correct orientation for expression. Thus aSalmonella or Shigella strain, as described above, may be used in thisfashion. Host cells suitable for use in the present invention willinclude but are not limited to Escherichia, Salmonella, Bacillus,Acinetobacter, Streptomyces, Methylobacter, Rhodococcus,Corynebacterium, Pseudomonas, Rhodobacter, and Synechocystis.

Particularly suitable in the present invention are members of theenteric class of bacteria. Enteric bacteria are members of the familyEnterobacteriaceae and include such members as Escherichia, Salmonella,and Shigella. They are gram-negative straight rods, 0.3-1.0×1.0-6.0 mm,motile by peritrichous flagella (except for Tatumella) or nonmotile.They grow in the presence and absence of oxygen and grow well onpeptone, meat extract, and (usually) MacConkey's media. Some grow onD-glucose as the sole source of carbon, whereas others require vitaminsand/or mineral(s). They are chemoorganotrophic with respiratory andfermentative metabolism but are not halophilic. Acid and often visiblegas is produced during fermentation of D-glucose, other carbohydrates,and polyhydroxyl alcohols. They are oxidase negative and, with theexception of Shigella dysenteriae 0 group 1 and Xenorhabdusnematophilus, catalase positive. Nitrate is reduced to nitrite (exceptby some strains of Erwinia and Yersina). The G+C content of DNA is 38-60mol % (T_(m), Bd). DNAs from species within most genera are at least 20%related to one another and to Escherichia coli, the type species of thefamily. Notable exceptions are species of Yersina, Proteus, Providenica,Hafnia and Edwardsiella, whose DNAs are 10-20% related to those ofspecies from other genera. Except for Erwinia chrysanthemi, all speciestested contain the enterobacterial common antigen (Bergy's Manual ofSystematic Bacteriology, D. H. Bergy et al., Baltimore: Williams andWilkins, 1984).

It is clear that host cells comprising the present regulator systems aresuitable for use in the invention. However, where it is desired to findnew strains having the present regulator systems, or to identify newregulator genes having greater functionality in non-native host cells,it will be possible to use the sequence information provided in theliterature and in this disclosure to identify and isolate such homologs.

Isolation of Homologs

A specific yhcS regulator has been identified in the E. coli genome andhas the nucleic acid sequence as set forth in SEQ ID NO:1. The promoterregion of the yhcRQP operon, responsive to the expression of this yhcSregulator has the nucleic acid sequence as set forth in SEQ ID NO:3. Itwill be apparent to the skilled artisan that homologs to the E. colisequences or others cited in the literature may easily be identifiedbased on current practices in molecular biology, and such homologs willbe equally applicable and useful in the present invention. For example,one of skill in the art may use the nucleic acid molecules of theinstant invention to isolate cDNAs and genes encoding a homologous YhcSprotein from the same or other bacterium species. Isolation ofhomologous genes using sequence-dependent protocols is well known in theart.

Examples of sequence-dependent protocols include, but are not limitedto, methods of nucleic acid hybridization, and methods of DNA and RNAamplification as exemplified by various uses of nucleic acidamplification technologies (e.g., PCR or ligase chain reaction).

For example, yhcS gene, either as cDNA or genomic DNA, could be isolateddirectly by using all or a portion of the instant nucleic acid moleculeas DNA hybridization probes to screen libraries from any desiredbacterium employing methodology well known to those skilled in the art.Specific oligonucleotide probes based upon the instant yhcS genesequence can be designed and synthesized by methods known in the art(Sambrook supra). Moreover, the entire sequences can be used directly tosynthesize DNA probes by methods known to the skilled artisan such asrandom primers DNA labeling, nick translation, or end-labelingtechniques, or RNA probes using available in vitro transcriptionsystems. In addition, specific primers can be designed and used toamplify a part of or full-length of the instant sequences. The resultingamplification products can be labeled directly during amplificationreactions or labeled after amplification reactions, and used as probesto isolate full length cDNA or genomic fragments under conditions ofappropriate stringency.

In addition, two short segments of the instant nucleic acid molecule maybe used in polymerase chain reaction protocols to amplify longer nucleicacid molecules encoding homologous yhcS genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid molecules wherein the sequence of one primer is derivedfrom the instant nucleic acid molecules, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor. Alternatively, the second primersequence may be based upon sequences derived from the cloning vector.For example, the skilled artisan can follow the RACE protocol (Frohmanet al., Proc. Natl. Acad. Sci. USA 85:8998-9002 (1988)) to generatecDNAs by using PCR to amplify copies of the region between a singlepoint in the transcript and the 3′ or 5′ end. Primers oriented in the 3′and 5′ directions can be designed from the instant sequences. Usingcommercially available 3′ RACE or 5′ RACE systems (Invitrogen, Carlsbad,Calif.), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al.,Proc. Natl. Acad. Sci. USA 86:5673-5677 (1989); Loh et al., Science243:217-220 (1989)). Products generated by the 3′ and 5′ RACE procedurescan be combined to generate full-length cDNAs (Frohman et al.,Techniques 1:165 (1989)).

Alternatively the yhcS sequences may be employed as an hybridizationreagent for the identification of homologs. The basic components of anucleic acid hybridization test include a probe, a sample suspected ofcontaining the gene or gene fragment of interest, and a specifichybridization method. Probes are typically single stranded nucleic acidsequences which are complementary to the nucleic acid sequences to bedetected. Probes are “hybridizable” to the nucleic acid sequence to bedetected. The probe length can vary from 5 bases to tens of thousands ofbases, and will depend upon the specific test to be done. Typically aprobe length of about 15 bases to about 30 bases is suitable. Only partof the probe molecule need be complementary to the nucleic acid sequenceto be detected. In addition, the complementarity between the probe andthe target sequence need not be perfect. Hybridization does occurbetween imperfectly complementary molecules with the result that acertain fraction of the bases in the hybridized region are not pairedwith the proper complementary base.

Hybridization methods are well defined. Typically the probe and samplemust be mixed under conditions which will permit nucleic acidhybridization. This involves contacting the probe and sample in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time that any possible hybridizationbetween the probe and sample nucleic acid may occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration theshorter the hybridization incubation time needed. Optionally achaotropic agent may be added. The chaotropic agent stabilizes nucleicacids by inhibiting nuclease activity. Furthermore, the chaotropic agentallows sensitive and stringent hybridization of short oligonucleotideprobes at room temperature (Van Ness and Chen, Nucl. Acids Res.19:5143-5151(1991)). Suitable chaotropic agents include guanidiniumchloride, guanidinium thiocyanate, sodium thiocyanate, lithiumtetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate,potassium iodide, and cesium trifluoroacetate, among others. Typically,the chaotropic agent will be present at a final concentration of about3M. If desired, one can add formamide to the hybridization mixture,typically 30-50% (v/v).

Various hybridization solutions can be employed. Typically, thesecomprise from about 20 to 60% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1M sodium chloride, about 0.05 to 0.1M buffers,such as sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9),about 0.05 to 0.2% detergent, such as sodium dodecylsulfate, or between0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kilodaltons),polyvinylpyrrolidone (about 250-500 kdal), and serum albumin. Alsoincluded in the typical hybridization solution will be unlabeled carriernucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA, e.g.,calf thymus or salmon sperm DNA, or yeast RNA, and optionally from about0.5 to 2% wt./vol. glycine. Other additives may also be included, suchas volume exclusion agents which include a variety of polarwater-soluble or swellable agents, such as polyethylene glycol, anionicpolymers such as polyacrylate or polymethylacrylate, and anionicsaccharidic polymers, such as dextran sulfate.

Nucleic acid hybridization is adaptable to a variety of assay formats.One of the most suitable is the sandwich assay format. The sandwichassay is particularly adaptable to hybridization under non-denaturingconditions. A primary component of a sandwich-type assay is a solidsupport. The solid support has adsorbed to it or covalently coupled toit immobilized nucleic acid probe that is unlabeled and complementary toone portion of the sequence.

Heterologous DNA Expression

Once a host cell comprising a yhcS regulator and the responsive yhcRQPoperon has been identified or constructed it will be necessary to insertthe heterologous nucleic acid molecule into the genome in the positionthat will be appropriate for expression. Methods for the transformationof microbial cells and integration of DNA into a genome are common andwell known in the art (see Sambrook supra). Typically a transformationvector is constructed for this purpose containing the essential elementsfor transformation and DNA integration. Typically the vector or cassettecontains sequences directing transcription and translation of therelevant gene, a selectable marker, and sequences allowing autonomousreplication or chromosomal integration. Suitable vectors comprise aregion 5′ of the gene which harbors transcriptional initiation controlsand a region 3′ of the DNA fragment which controls transcriptionaltermination. It is most preferred when both control regions are derivedfrom genes homologous to the transformed host cell, although it is to beunderstood that such control regions need not be derived from the genesnative to the specific species chosen as a production host.

Heterologous nucleic acid molecules suitable for expression by themethods of the invention are virtually unlimited. In some instances theheterologous nucleic acid molecule may not encode a protein and beexpressed for the purpose of controlling or suppressing (as in antisenseorientation for example) other genetic elements in the genome. Morecommonly, the foreign DNA will encode a protein and typically an enzymethat is part of a pathway. It will be appreciated that a single DNAfragment may be expressed by the present method or several linkedfragments comprising all or a part of an enzymatic pathway. It istherefore within the scope of the present invention to expressheterologous nucleic acid molecules encoding at least one proteinwherein the at least one protein is part of an enzymatic biosyntheticpathway producing a product selected from the group consisting ofisoprenoids; terpenoids, tetrapyrroles, polyketides, vitamins, aminoacids, fatty acids, proteins, nucleic acids, carbohydrates,antimicrobial agents, and anticancer agents.

In some instances it will be useful to monitor the expression oractivation of the regulator systems through the use of a reporter.Reporters are common and well known in the art and a non-inclusive listof those suitable in the present invention are luxCDABE, bgaB, cat,dsRed, galK, gfp, lacZ, luc, luxAB, nptll, phoA, uidA, and xylE.

One of the key advantages of the present invention is the ability tocontrol the expression of the heterologous DNA by the action of aninducer. Applicant's discovery that the present regulator genes areresponsive to aromatic carboxylic acids is fortuitous in that thesecompounds are inexpensive and easily obtained. Any aromatic carboxylicacid will have utility in the present method where para-hydroxybenzoicacid, para-hydroxycinnamic acid, cinnamic acid, salicylic acid, benzoicacid, and 1-napthoic acid are preferred.

Description of the Preferred Embodiments

As described in the following examples, during the course of LuxArrayand DNA array analyses. Applicants discovered that expression of an E.coli operon, yhcRQP, was highly induced by treatment of E. coli cellswith aromatic carboxylic acids, such a para-hydroxybenzoic acid (pHBA),para-hydroxycinnamic acid (pHCA), and cinnamic acid (CA). A luxCDABEgene fusion with the promoter region of this operon controlling thebioluminescent reporter was used to characterize expression of thisoperon. In the absence of inducing molecules the expression level wasvery low, just slightly above background. However, when the cell wastreated with inducer, the expression level was dramatically elevated,where expression increased nearly 1000-fold. This represents much higherexpression than found in typical LysR family members. Furthermore, theexpression level was dependent on the concentration of inducer added.

The gene immediately upstream of the yhcRQP operon at open reading frameb3243, YhcS, is a putative member of the LysR family of transcriptionregulators. Using a transposon insertion mutation in yhcS, expression ofthe yhcRQP-luxCDABE gene fusion was no longer induced by aromaticcarboxylic acids. Thus, it was shown that yhcS encodes a positive-actingtranscriptional regulator responsible for the dramatic, tunable changesin gene expression of the yhcRQP operon.

This promoter/regulator system can be used to control and regulateexpression of other genes and operons of interest by applying standardmolecular biology methods. Furthermore, by analogy to other LysR familyproteins, the YhcS protein likely binds to these aromatic carboxylicacid molecules and changes conformation such that it activates geneexpression upon DNA binding. This conformation change may be useful tosense small molecules in nano-scale systems.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees in Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usage and conditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by Sambrook, J.,Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989); byT. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with GeneFusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984);and by Ausubel, F. M. et al., Current Protocols in Molecular Biology,pub. by Greene Publishing Assoc. and Wiley-Interscience, Hoboken, N.J.(1987).

Standard genetic methods for transduction used in the Examples are wellknown in the art and are described by Miller, J. H., Experiments inMolecular Genetics, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1972).

The meaning of abbreviations is as follows: “kb” means kilobase(s), “hr”means hour(s), “min” means minute(s), “sec” means second(s), “d” meansday(s), “ml” means milliliter(s), “μl” means microliter(s), “nl” meansnanoliter(s), “μg” means microgram(s), “ng” means nanogram(s), “mM”means millimolar, “μM” means micromolar, “nm” means nanometer(s),“OD₆₀₀” means the optical density measured at a wavelength of 600 nm,“RLU” means relative light units.

Media and Culture Conditions:

Materials and methods suitable for the maintenance and growth ofbacterial cultures were found in Experiments in Molecular Genetics(Jeffrey H. Miller), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1972); Manual of Methods for General Bacteriology (PhillipGerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, WillisA. Wood, Noel R. Krieg and G. Briggs Phillips, eds), pp. 210-213,American Society for Microbiology, Washington, D.C. (1981); or Thomas D.Brock in Biotechnology: A Textbook of Industrial Microbiology, SecondEdition (1989) Sinauer Associates, Inc., Sunderland, Mass. All reagentsand materials used for the growth and maintenance of bacterial cellswere obtained from Aldrich Chemicals (Milwaukee, Wis.), BD DiagnosticSystems (Sparks, Md.), Invitrogen Corp. (Carlsbad, Calif.), or SigmaChemical Company (St. Louis, Mo.) unless otherwise specified.

LB medium contains the following per liter of medium: Bacto-tryptone (10g), Bacto-yeast extract (5 g), and NaCl (10 g).

Vogel-Bonner medium contains the following per liter: 0.2 g MgSO₄.7H₂O,2 g citric acid.1H₂O, 10 g K₂HPO₄ and 3.5 g NaNH₄HPO₄.4H₂O.

Minimal M9 medium contains the following per liter of medium: Na₂HPO₄ (6g), KH₂PO₄ (3 g), NaCl (0.5 g), and NH₄Cl (1 g).

Above media were autoclaved for sterilization then 10 ml of 0.01 M CaCl₂and 1 ml of 1 M MgSO₄.7H₂O were added to M9 medium. Vitamin B1 (thiamin)was added at 0.0001% to both Vogel-Bonner and M9 media. Carbon sourceand other nutrients and supplements were added as mentioned in theExamples. All additions were pre-sterilized before they were added tothe media.

Molecular Biology Techniques:

Restriction enzyme digestions, ligations, transformations, and methodsfor agarose gel electrophoresis were performed as described in Sambrooksupra. Polymerase Chain Reactions (PCR) techniques were found in White,B., PCR Protocols: Current Methods and Applications, Volume 15 (1993)Humana Press Inc, Totowa, N.J.

Example 1 Gene Expression Profiling of Para-Hydroxybenzoate-Treated E.coli Cells

The alterations in the E. coli gene expression profile upon exposure topHBA were examined using DNA microarray technology. E. coli strain DE112(Van Dyk et al. Appl. Environ. Microbiol. 60:1414-1420 (1994)) was grownin Vogel-Bonner minimal medium with glucose as a carbon source to anOD₆₀₀ of 0.2. At this point the culture was split in two flasks and pHBAin the acid form was added to one flask from a stock solution in ethanolto achieve a final pHBA concentration of 25 mM. The pH of the medium inthe flask with pHBA was lowered by an unmeasured amount. An equivalentvolume of ethanol without pHBA was added to the other flask.Approximately 58% growth inhibition resulted from the pHBA treatmentunder these conditions. Cells were harvested from the control andtreated flasks at 30 and 60 minutes after pHBA addition. RNA isolation,array hybridization, and data analysis were done as previously described(Wei et al, J. Bacteriol. 183:545-556 (2001), Smulski et al. J.Bacteriol. 183:3353-3364 (2001)). Among the genes that were highlyinduced by this treatment at the 60 minute time point were the yhcR,yhcQ, and yhcP genes (Table 1). The experiment was repeated, and again,these three genes were highly upregulated (Table 2). These three genesare predicted to be transcribed as an operon. The reproducible observedco-regulation in response to pHBA treatment is consistent with thisprediction. Thus, these three genes will be referred to as the yhcRQPoperon.

TABLE 1 Upregulation of genes in the yhcRQP operon after 60 minutes pHBAtreatment, experiment 1 Blattner Signal in Signal in Ratio Gene No.*untreated pHBA treated (treated/untreated) yhcR b3242 0.0190 1.37 72.3yhcQ b3241 0.393 1.61 4.09 yhcP b3240 1.17 7.52 6.45

TABLE 2 Upregulation of genes in the yhcRQP operon after 60 minutes pHBAtreatment, experiment 2 Blattner Signal in Signal in pHBA Ratio GeneNo.* untreated treated (treated/untreated) yhcR b3242 0.687 7.18 10.5yhcQ b3241 0.270 6.02 22.3 yhcP b3240 0.994 12.0 12.1 *Blattner et al.Science 277: 1453-1462 (1997).

Example 2 LuxArray Analysis of Para-Hydroxycinnamic Acid and CinnamicAcid Treated E. coli

Gene expression profiles were done using LuxArray version 1.04, whichhas been fully described (Van Dyk et al. J. Bacteriol. 183:5496-5505(2001), Gonye et al. U.S. Patent Application Publication 20030219736).This method utilizes a set of bioluminescent gene fusions to ⅓ of E.coli transcriptional units in a tolC host strain that is hypersensitiveto many compounds, including pHCA. Sublethal concentrations of pHCA, 10mM and 5 mM, and CA, 8 mM and 4 mM, at which stress and other responsescan be detected using luxCDABE reporter gene fusions, were used. Each ofthese treatments yielded nearly identical expression patterns,suggesting a similar cellular response to these two aromatic compounds.A predominant feature observed in both these profiles was that one genefusion, lux-a.pk035.c7, was the several hundred-fold upregulated. Thelux-a.pk035.c7 gene fusion contains an E. coli chromosomal segmentbetween nucleotides 3385829 and 3386761 according to the E. coli genomicsequence, which contains the promoter region of the putative yhcRQPoperon and the entire yhcR gene and the 5′ end of the yhcQ gene. Thischromosomal segment is joined to the luxCDABE gene fusion in theparental plasmid, pDEW201 (Gonye et al. U.S. Patent ApplicationPublication 20030219736) thus forming junction between yhcQ gene andluxC. Accordingly, this gene fusion will report on expression of theyhcQ gene and any other genes cotranscribed with it. As detailed inExample 1, it is likely that yhcR, yhcQ, and yhcP form an operon, thusthis gene fusion is referred to as a yhcRQP-luxCDABE gene fusion. Thestrain containing this gene fusion was given the name DPD2411, and theplasmid within this strain that contains yhcRQP-luxCDABE gene fusion wascalled pDEW655.

Table 3 shows the bioluminescent response of this reporter gene fusionto pHCA, CA, and ethanol. As part of a larger LuxArray experiment, twoindependent, actively growing, cultures carrying the yhcRQP-luxCDABEgene fusion in LB medium were each split three ways at time zero. Twoaliquots were treated with different concentrations of each chemical andthe third aliquot was the untreated control. The normalizedbioluminescent signal from each of two replicas in the LuxArray is shownfor the measurements at four time points (in minutes).

The dramatic upregulation of expression in response to pHCA and CAtreatments at each of the time points other than the initial, zero timepoint is clear. In contrast, ethanol treatment does not induce increasedbioluminescence.

TABLE 3 Responses of the yhcRQP-luxCDABE gene fusion to pHCA, CA, orethanol Replica 1 Replica 2 normalized RLU normalized RLU Treatment 0min 45 min 90 min 135 min 0 min 45 min 90 min 135 min  0 mM pHCA 0.0610.071 0.067 0.039 0.043 0.04 0.039 0.022  5 mM pHCA 0.056 10.695 14.27613.533 0.043 9.325 14.754 10.587 10 mM pHCA 0.056 9.094 14.314 15.2830.034 7.31 11.164 12.867  0 mM CA 0.03 0.034 0.024 0.013 0.08 0.0520.032 0.015  4 mM CA 0.026 4.092 5.845 5.394 0.062 2.928 4.951 3.678  8mM CA 0.022 4.937 8.379 6.981 0.054 3.405 6.193 5.746 0% ethanol 0.0210.023 0.017 0.009 0.045 0.028 0.018 0.008 3% ethanol 0.024 0.024 0.0220.012 0.038 0.029 0.026 0.014 5% ethanol 0.022 0.014 0.022 0.015 0.0370.017 0.017 0.014

Example 3 Regulation of yhcRQP Expression by YhcS

The yhcS gene of E. coli encodes an uncharacterized member of the LysRfamily of positive acting regulatory molecules. This gene is locatedimmediately adjacent to the yhcRQP operon that was found to beupregulated by pHBA treatment in DNA array experiments and by pHCA andCA treatments in LuxArray experiments. The possibility that YhcScontrols expression of yhcRQP was tested using a yhcS null mutation.

Such a mutation was found in an E. coli library of transposon insertionmutations constructed using the transposome system based on the Tn5transposon (Epicentre, Madison, Wis.). A transposome is a protein-DNAcomplex composed of the EZ::TN<Kan-1> transposon and the EZ::TNtransposase. The EZ::TN transposase is bound to the ends of thetransposon, which facilitates the formation of a stable synapticcomplex. The transposome requires Mg⁺² to initiate the insertion of theEZ::TN<Kan-1> transposon into target DNA. The cellular levels of Mg⁺²are sufficient to activate the transposome. Thus, the electroporation ofthe transposome into cells permits the in vivo insertion of theEZ::TN<Kan-1> transposon into bacterial genomes.

The EZ::TN<Kan-1> transposome was electroporated into electroporationcompetent E. coli strain DH5αE cells (Invitrogen, Carlsbad, Calif.).Following electroporation, the cells were grown in SOC medium(Initrogen) for one hour at 37° C. with aeration. Subsequently, thecells were plated onto LB agar plates containing kanamycin (50 μg/ml)(LB+Kan) and incubated overnight at 37° C. Individual colonies wereinoculated into 96-well microtiter plates containing 150 μl of LB+Kanand incubated overnight at 37° C.

“Single Primer PCR” was used to determine the identity of each E. colitransposon mutation. Using a single DNA primer that was complementary toone end of the EZ::TN<Kan-1> transposon, PCR products were generated.Subsequently, a second DNA primer (located internal and adjacent to thePCR primer) was used to sequence the PCR products. The DNA primer usedin the PCR reaction was either Kan-2FP(PCR) (SEQ ID NO:4) orKan-2RP(PCR) (SEQ ID NO:5) and the DNA primer used for DNA sequencingwas either Kan-2FP(PCR) (SEQ ID NO:4) or Kan-2RP(PCR) (SEQ ID NO:5),respectively. The PCR reaction conditions were the following: (1) 94°C., 15 minutes (2) 20 cycles—94° C., 30 seconds; 60° C., 30 seconds; 72°C., 3 minutes (3) 30 cycles—94° C., 30 seconds; 40° C., 30 seconds; 72°C., 2 minutes (4) 30 cycles—94° C., 30 seconds; 60° C., 30 seconds; 72°C., 2 minutes (5) 72° C., 7 minutes. The PCR reactions were prepared forDNA sequencing using the QIAquick PCR Purification Kit (Qiagen,Valencia, Calif.).

The yhcS transposon mutant was identified using PCR amplification primerKan-2FP(PCR) (SEQ ID NO:4) and DNA sequencing primer Kan-2FP-1 (SEQ IDNO:6). The transposon mutation was confirmed using gene-specificprimers: YhcS.F (SEQ ID NO:8) and YhcS.R (SEQ ID NO:9) andtransposon-specific primers Kan-2FP-1 (SEQ ID NO:6) and Kan-2RP-1 (SEQID NO:7).

The size of the yhcS gene is ˜929 base pairs. The transposon insertionsite within the yhcS gene is ˜330 base pairs away from the 5′ end ofyhcS. A PCR reaction done with the YhcS.F and Kan-2RP-1 primers yieldeda PCR fragment ˜550 base pairs and PCR primers YhcS.R and Kan-2FP-1yielded a PCR product <400 base pairs in size.

E. coli strain DPD2410 is the DH5αE derived strain containing theyhcS::TN<Kan> mutation. Strains DH5αE and DPD2410 were transformed withpDEW655 to generate E. coli strains DPD2413 and DPD2415, respectively. Asingle colony of each of these two strains from an LB plate containing150 μg/ml Ampicillin was used to inoculate 200 μl LB medium in wells ofa 96 well, white microplate (Microlite, Dynex Technologies, Chantilly,Va.). The plate was incubated for 90 minutes at 37° C.; then 50 μl ofthe cultures was added to 50 μl of LB medium or to 50 μl of LB mediumcontaining pHBA in the acid form, which had been added from a stocksolution in ethanol. The final concentration of pHBA was 5 mM and thefinal concentration of ethanol was 0.25%. The pH of the medium with pHBAwas lowered by an unmeasured amount. The bioluminescence was quantitatedwith a Luminoskan Ascent microplate luminometer (Thermo Labsystems,Franklin, Mass.). The results of this study are presented in FIG. 1,which is a plot of the bioluminescence intensity in relative light units(RLU) versus time in minutes. In the Figure, addition of pHBA was madeat time zero. Solid lines are the response in the yhcS⁺ strain, DPD2413.Dotted lines are the response in the yhcS⁻ strain, DPD2415. Circlesrepresent pHBA treated cultures and triangles represent untreatedcultures. FIG. 1 clearly shows that pHBA induced rapid and dramaticupregulation of the yhcRQP-luxCDABE expression in the yhcS⁺ host strain,but that this upregulation was essentially abolished in theyhcS::TN<Kan> host strain.

A derivative of E. coli strain MG1655 (obtained from Prof. Douglas Berg,Washington University School of Medicine, St. Louis, Mich.) with theyhcS::TN<Kan> mutation was made by P1clr100Cm mediated transductionusing phage grown on strain DPD2410 as a donor and selection forkanamycin resistance. The presence of the yhcS::TN<Kan> mutation in oneof the resultant transductants, named DPD2433, was confirmed by PCRamplification. Plasmid pDEW655 was moved to E. coli strains MG1655 andDPD2433 by transformation, selecting for Ampicillin resistance togenerate strains DPD2436 and DPD2437, respectively. The bioluminescentresponse of these two strains to pHBA was tested. Aliquots (50 μl) ofactively growing cultures at 37° C. in LB medium that had beenpreviously diluted, and from overnight cultures in LB medium with 150μg/ml Ampicillin were added to 50 μl of LB medium at pH 7.0 containingpHBA as the sodium salt form. Several concentrations of pHBA weretested. Table 4 below shows the response in these two host strains atthirty minutes after cells were added to pHBA containing medium.

TABLE 4 Bioluminescence response of the yhcRQP-luxCDABE gene fusion[pHBA], RLU Ratio treated/control mM yhcS+ yhcS− yhcS+ yhcS− 100 0.4370.045 0.693 0.055 50 91.7 0.614 145 0.753 25 66.6 1.59 106 1.95 12.530.8 1.82 48.8 2.23 6.2 16.2 1.42 25.7 1.75 3.1 10.2 1.16 16.2 1.42 1.66.72 1.02 10.6 1.25 0 0.631 0.815 1 1

The yhcS::TN<Kan> mutation almost completely eliminated the upregulationof expression induced by pHBA treatment at all concentrations tested.Also note that in the yhcS⁺ strain, the level of gene expression asquantitated by the degree of bioluminescence varied with theconcentration of pHBA added. Thus, the amount of inducer added can beused to tune the expression level from this promoter.

Overall, in two different E. coli host strains, a functional YhcS wasrequired for upregulation of yhcRQP expression in response to pHBAaddition. These results prove that YhcS is a positive acting factor forupregulation of transcription of the yhcRQP operon.

Example 4 Structure Activity Relationships for YhcS Activation

Further characterization of the signals that trigger activation of YhcS,was done using the yhcRQP-luxCDABE gene fusion containing E. colistrains DPD2411 or DPD2436. Table 5 shows the results of bioluminescenceactivation tests done with cells in LB medium at pH 7.0, as described inExample 3. Several weak, aromatic acid molecules in addition to thoseshown in the Examples above activated expression. Thus, the knowninducing molecules comprise pHBA, pHCA, CA, salicylate, benzoate, and1-napthoate.

TABLE 5 Upregulation of yhcRQP-luxCDABE expression by aromaticcarboxylic acids Ex- Concen- peri- tration ment E. coli of maximumtreated control code* strain Compound response RLU RLU Ratio A DPD24111- 2.5 mM  29.662 0.2244 132 napthoate A DPD2411 Sodium 25 mM 69.6780.231 302 pHBA B DPD2436 Sodium 6.2 mM  3.759 0.2898 13 benzoate CDPD2436 Sodium 12.5 mM   8.093 0.6792 12 benzoate C DPD2436 Sodium 50 mM91.658 0.6311 145 pHBA D DPD2436 Sodium 6.2 mM  14.703 0.1908 77salicylate D DPD2436 Sodium 50 mM 48.643 0.196 248 pHBA *Experimentswith the same code letter were done simultaneously using one split E.coli culture.

Compounds tested that did not induce expression were defined as thosefor which there resulted less than 3-fold increase in light productionfrom E. coli strains containing pDEW655. Compounds unrelated instructure to the known inducing molecules did not induce expression.Those tested were acetate, propionate, ethanol, limonene, NaCl,polymyxin sulfate, benzalkonium chloride, gramicidin S, and SDS. Inaddition, several compounds related in structure to the inducingmolecules were not inducers, including methyl paraben,p-hydroxystryrene, 2-biphenylcarboxylate, and L-tyrosine. Thus, therequirement for the carboxylate moiety was demonstrated by the lack ofresponse to methyl paraben, the methyl ester of pHBA, and top-hydroxystryrene, a molecule related to pHCA but lacking thecarboxylate group. The requirement for an aromatic ring was demonstratedby the lack of response to non-aromatic carboxylic acids, acetate andpropionate.

The response of this regulatory system is specific for certain aromaticcarboxylic acids. This class of molecules includes compounds that areenvironmentally friendly and relatively inexpensive, such as sodiumbenzoate.

Example 5 Internal Acidification is not the Signal that Activates YhcS

All characterized activators of YhcS are weak acids such as pHBA andpHCA. Thus, the inducing condition could potentially be eitheracidification of the cytoplasm or presence of the conjugate molecule.The fact that non-aromatic weak acids propionate and acetate, which areknown to cause cytoplasmic acidification, did not induce expression ofthe yhcRQP-luxCDABE gene fusion suggested that cytoplasmic acidificationwas not the inducing signal. This conclusion was confirmed byexperiments comparing upregulation of yhcRQP-luxCDABE mediated by YhcSto that mediated by other well-known acidification responsive regulatorycircuits. Three E. coli strains, each in the same host strain butcarrying different, plasmid-borne, promoter-luxCDABE fusions were used.Strain DPD2411 contains a yhcRQP-luxCDABE gene fusion as described inExample 2. Strain DPD2084 contains a yciG-luxCDABE gene fusion that hasbeen previously described. Strain DPD3282 contains a lysU-luxCDABE genefusion that was part of the LuxA collection of gene fusions, describedin Example 2. The plasmid in this strain, pDEW558, contains an E. colichromosomal segment between nucleotides 4350990 and 4353107 according tothe E. coli genomic sequence; the orientation of the chromosomal segmentis such that the lysU promoter region controls expression of luxCDABE.Each of these three strains was grown overnight in Vogel-Bonner minimalmedium with 0.4% glucose as the carbon source and supplemented withL-proline, L-lysine, uracil and 25 μg/ml Ampicillin. The overnightcultures were diluted into the same medium except lacking Ampicillin andincubated at 37° C. until in mid-exponential growth. Aliquots (50 μl) ofthese actively growing cultures were added to 50 μl of the same mediumat pH 7.0 without Ampicillin but containing various concentrations ofsodium acetate or sodium salicylate in the wells of a 96 well, whitemicroplate. (Microlite, Dynex Technologies). Immediately after addingthe cell culture, the bioluminescence was quantitated in a microplateluminometer in the kinetic mode. Table 6 shows the results at 100minutes after acetate addition or salicylate addition. Treatment of E.coli with acetate did not activate expression of yhcRQP-luxCDABE, butdid activate the other two acid responsive regulatory circuits.Conversely, addition of sodium salicylate upregulated expression ofyhcRQP-luxCDABE, but did not increase expression of the other two acidresponsive gene fusions at the concentrations tested. Thus, it can beconcluded that YhcS is not responding to acidification signals, butrather is responding to the presence of the aromatic molecules.

TABLE 6 Comparison of acid inducible gene fusions and yhcRQP- luxCDABEresponses to acetate and salicylate RLU at 100 minutes 0 mM 0.6 mM 5.0mM Gene 0 mM 80 mM 160 mM salic- salic- salic- fusion acetate acetateacetate ylate ylate ylate yhcRQP- 0.13 0.14 0.08 0.32 7.9 26.0 luxCDABEyciG- 0.19 0.35 0.80 0.18 0.19 0.13 luxCDABE lysU- 1.3 1.4 6.0 1.3 1.30.89 luxCDABE

1. A method for the inducible expression of a heterologous nucleic acidmolecule comprising: a) providing a host cell having a genomecomprising: i) a yhcS regulator gene responsive to an aromaticcarboxylic acid inducer; ii) a promoter region, responsive to expressionof the yhcS regulator gene; and iii) at least one heterologous nucleicacid molecule; wherein the at least one heterologous nucleic acidmolecule is operably linked to the promoter region; b) contacting thehost cell of (a) with an aromatic carboxylic acid inducer wherein the atleast one heterologus nucleic acid molecule is expressed.
 2. A methodaccording to claim 1 wherein the at least one heterologous nucleic acidmolecule encodes at least one protein.
 3. A method according to claim 2wherein the at least one protein is part of an enzymatic biosyntheticpathway producing a product selected from the group consisting ofisoprenoids, terpenoids, tetrapyrroles, polyketides, vitamins, aminoacids, fatty acids, proteins, nucleic acids, carbohydrates,antimicrobial agents, and anticancer agents.
 4. A method according toclaim 1 wherein the at least one heterologous nucleic acid moleculeencodes a reporter.
 5. A method according to claim 4 wherein thereporter is selected from the group consisting of luxCDABE, bgaB, cat,dsRed, galK, gfp, lacZ, luc, luxAB, nptII, phoA, uidA, and xylE.
 6. Amethod according to claim 1 wherein the aromatic carboxylic acid induceris selected from the group consisting of para-hydroxybenzoic acid,para-hydroxycinnamic acid, cinnamic acid, salicylic acid, benzoic acid,and 1-napthoic acid.
 7. A method according to claim 1 wherein thepromoter region, responsive to expression of the yhcS regulator gene ispromoter region isolated from the yhcRQP operon.
 8. A method accordingto claim 1 wherein the host cell is an enteric bacteria.
 9. A methodaccording to claim 1 wherein the host cell is selected from the group ofgenera consisting of Escherichia, Salmonella, Bacillus, Acinetobacter,Streptomyces, Methylobacter, Rhodococcus, Corynebacterium, Pseudomonas,Rhodobacter, and Synechocystis.
 10. A method for the inducibleexpression of a heterologous nucleic acid molecule comprising: a)providing an enteric bacterial host cell having a genome comprising: i)a yhcS regulator gene responsive to an aromatic carboxylic acid inducer;ii) a promoter region, responsive to expression of the yhcS regulatorgene; and iii) at least one heterologous nucleic acid molecule; whereinthe at least one heterologous nucleic acid molecule is operably linkedto the promoter region; b) contacting the host cell of(a) with anaromatic carboxylic acid inducer wherein the at least one heterologusnucleic acid molecule is expressed.
 11. A method according to claim 10wherein the yhcS regulator gene responsive to an aromatic carboxylicacid inducer is an isolated nucleic acid molecule selected from thegroup consisting of: a) an isolated nucleic acid molecule comprisingnucleic acid sequence SEQ ID NO:1; and b) an isolated nucleic acidmolecule, which hybridizes to SEQ ID NO:1 after being washed with0.1×SSC, 0.1% SDS at 65° C. and washed with 2×SSC, 0.1% SDS followed bya second wash in 0.2×SSC, 0.1% SDS.
 12. A method according to claim 10wherein the promoter region, responsive to expression of the yhcSregulator gene is an isolated nucleic acid molecule selected from thegroup consisting of: a) an isolated nucleic acid molecule comprisingnucleic acid sequence SEQ ID NO:3; and b) an isolated nucleic acidmolecule, which hybridizes to SEQ ID NO:3 after being washed with0.1×SSC, 0.1% SDS at 65° C. and washed with 2×SSC, 0.1% SDS followed bya second wash in 0.2×SSC, 0.1% SDS.
 13. A method according to claim 10wherein the enteric bacterial host cell is E. coli.